Identification of an Intermediate Form of Ferredoxin That Binds Only Iron Suggests That Conversion to Holo-Ferredoxin Is Independent of the ISC System in Escherichia coli

Abstract

Escherichia coli [2Fe-2S]-ferredoxin and other ISC proteins encoded by the iscRSUA-hscBA-fdx-iscX (isc) operon are responsible for the assembly of iron-sulfur clusters. It is proposed that ferredoxin (Fdx) donates electrons from its reduced [2Fe-2S] center to iron-sulfur cluster biogenesis reactions. However, the underlying mechanisms of the [2Fe-2S] cluster assembly in Fdx remain elusive. Here, we report that Fdx preferentially binds iron, but not the [2Fe-2S] cluster under cold-stress conditions (≤ 16°C). The iron binding in Fdx is characterized by a unique absorption peak at 320 nm based on UV-visible spectroscopy. In addition, the iron-bound form of Fdx could be converted to the [2Fe-2S] cluster bound form after transferring cold-stressed cells to normal cultivation temperatures above 25°C. In vitro experiments also revealed that Fdx could utilize bound iron to assemble [2Fe-2S] cluster by itself. Furthermore, inactivation of the genes encoding IscS, IscU, and IscA did not limit [2Fe-2S] cluster assembly in Fdx, which was also observed by inactivating the isc or suf operon, indicating that iron-sulfur cluster biogenesis in Fdx arose from a unique pathway in E. coli. Our results suggest that the intracellular assembly of [2Fe-2S] clusters in Fdx is susceptible to the environmental temperatures. Iron binding form of Fdx (Fe-Fdx) is a precursor during its maturation to a cluster binding form ([2Fe-2S]-Fdx), and self-assembly of the [2Fe-2S] clusters during temperature increases is not strictly reliant on other specific iron donors and scaffold proteins within the Isc or Suf systems. Importance Fdx is an electron carrier that is required for the maturation of many other iron-sulfur proteins. Its function strictly depends on its [2Fe-2S] center that bonds with the cysteinyl S atoms of four cysteine residues within Fdx. However, the assembly mechanism of the [2Fe-2S] clusters in Fdx remains controversial. This study reports that Fdx fails to form its [2Fe-2S] cluster under cold stress-conditions, but instead binds a single Fe atom at the cluster binding site. Moreover, when temperatures increase, Fdx can assemble clusters by itself from its iron-only binding form in E. coli cells. A possibility remains that Fdx can effectively accept clusters from multiple sources. Nevertheless, our results suggest that Fdx has a strong iron binding activity that contributes to the assembly of its own [2Fe-2S] cluster and that Fdx may act as a temperature sensor to regulate Isc system-mediated iron-sulfur cluster biogenesis.

Establishing Efficient Bisphenol A Degradation by Engineering Shewanella oneidensis

“…E. coli [2Fe-2S] ferredoxin and other iron–sulfur proteins encoded by the iscRSUA - hscBA - fdx - iscX operon are responsible for the assembly of the iron–sulfur cluster. Ferredoxin contributes electrons from the [2Fe-2S] center of its reduction to the formation of iron–sulfur clusters . Moreover, [2Fe-2S] ferredoxin genes, including So0747 and So2269 , have also been identified in Shewanella . ,, Therefore, our results (Figure A) imply that various [2Fe-2S] ferredoxins might substitute for Fd bisd in the P450 bisd monooxygenase system.…”

“…Ferredoxin contributes electrons from the [2Fe-2S] center of its reduction to the formation of iron−sulfur clusters. 66 Moreover, [2Fe-2S] ferredoxin genes, including So0747 and So2269, have also been identified in Shewanella. 52,67,68 Therefore, our results (Figure 4A) imply that various [2Fe-2S] ferredoxins might substitute for Fd bisd in the P450 bisd monooxygenase system.…”

Bacterioferritin: a key iron storage modulator that affects strain growth and butenyl-spinosyn biosynthesis in Saccharopolyspora pogona

“…As expected, the expression level of Bfr was significantly higher in :: bfr than in the wild-type, and it failed to be detected in Δ bfr . In addition, the expression level of ferredoxin, which is involved in cellular iron binding and electron transfer in redox reactions [ 37 ], was also significantly higher in :: bfr . Notably, the expression levels of thioredoxin reductase (TrxB), catalase-peroxidase (KatG) and superoxide dismutase (SOD), which are involved in oxidative stress response [ 38 – 40 ], were all significantly increased in :: bfr , suggesting a possible difference in antioxidant capacity between the mutant and wild-type strains (Table 1 ).…”

“…5 ). In addition, ferredoxin is a Fe-S protein that mediates electron transfer in a variety of metabolic reactions, aid in iron storage and utilization, and facilitate related metabolism in :: bfr [ 37 ]. Fe 2+ is oxidized to Fe 3+ and stored in Bfr, and then reduced to Fe 2+ when needed.…”

Roles of conserved active site residues in the IscS cysteine desulfurase reaction

“…Buffer A was used as the negative control and endonuclease III (Nth) was used as the positive control. The iron content in E. coli Nth was calculated from the iron-ferrozine determination, as previously described by Ren et al (2021) , and because E. coli Nth contains a stable [4Fe-4S] cluster, the protein should have equal amounts of iron and sulphur. Therefore, the extinction coefficient of sulphur was calculated based on the content of iron in Nth.…”